Elevated hydrostatic pressure disturbs expression of growth factors in human renal epithelial cells

Obstructive uropathy is a common kidney disease caused by elevated hydrostatic pressure (HP), but relevant molecular and cellular mechanisms have not yet been well understood. In this study, we ex vivo investigated the effects of elevated HP on human renal epithelial cells (HREpCs). Primary HREpCs were subjected to 100 cmH2O HP for 8 or 48 h. Then, the cells were cultured without HP stimulation for another 24 h or 72 h. Cell morphology showed almost no change after 8h HP treatment, but exhibited reversible elongation after 48h HP treatment. HP treatment for 8 h increased the expression of TGFB1 and VEGFA but decreased the expression of CSF2 and TGFB2. On the other hand, HP treatment for 48 h downregulated the expression of CSF2, TGFB2, PDGFB, VEGFA, and VEGFB, while upregulated the expression of TGFB3. Interestingly, all changes induced by 48 h HP treatment were detected more severe compared to 8 h HP treatment. In conclusion, elongated ex vivo HP loading to renal epithelial cells induces reversible changes on cell morphology and disturbs the expression of several growth factors, which provides novel mechanistic insight on elevated HP-caused kidney injury such as obstructive uropathy.


Introduction
The kidneys are pivotal to maintaining the fluid and electrolyte balance.Obstructive uropathy is most commonly caused by malignancy, urolithiasis, and others [1].Acute kidney injury (AKI) was present in over 70% of the patients with obstructive uropathy [1].In obstructed human kidneys, the intrarenal pressures (IRPs) increase from a few cmH 2 O to more than 90 cmH 2 O [2].Animal studies have proved that elevated IRPs can damage kidneys [3][4][5].In a porcine study, hydrostatic pressure (HP) induces renal cellular injury within a few hours at IRPs of 20 cmH 2 O or greater [3].Histological analysis also shows that elevated HP leads to marked vacuolization and degeneration in renal tubules [4,5].Additionally, the release of the ureteral obstruction leads to either complete or partial reversal of kidney injury, contingent upon the severity and duration of the obstruction [6,7].
The effects of HP on many cell types, including hepatic stellate cells, renal cells, bladder fibroblasts, and cancer cells, have been investigated [8][9][10][11][12][13].These studies show that HP can increase cell proliferation, cell detachment, tissue fibrosis, epithelial-mesenchymal transition (EMT), and cancer metastasis [8][9][10][11][12][13].However, the effects of HP on renal epithelial cells have rarely been investigated.One study has shown that in vitro exposure of renal cells to 60 cmH 2 O HP results in increased cell number and rearranged microfilament structure [11].Using a unilateral ureteral obstruction mouse model and primary human renal epithelial cells (HREpCs), we also found that elevated HP can induce renal fibrosis and EMT of renal epithelial cells [12,14].
It has been demonstrated that various growth factors maintain kidney physiological functions and are involved in the pathogenesis of kidney disease [15].In this study, we investigated how elevated HP affected the expression of growth factors in the HREpCs, and further interested to know whether the elevated HP-induced changes are reversible.

Cell culture
HREpCs were purchased from ScienCell Research Laboratories (#4120, ScienCell).Cells were maintained in an epithelial cell medium (#4101, ScienCell) and cultured at 37˚C in a humidified atmosphere of 5% CO 2 and 95% air.

HP loading
We used a pneumatic pressurizing system (Strex.Inc) to mimic mechanical stimuli of kidney cells experienced by obstructive uropathy.Briefly, 5 × 10 5 HREpCs were seeded in 6 cm diameter culture dishes.After an overnight incubation, the cells were placed inside the pressure chamber.Then, the chamber was placed inside a CO 2 incubator and connected to the control box outside the incubator via vacuum tubing.Cells were loaded to 100 cmH 2 O sustained HP for 8 or 48 h.To ensure cell viability, a 30 s gas exchange occurs every 60 min by releasing pressure and re-pressurizing the chamber.
After 8h (short-term) or 48h (long-term) treatment with 100 cmH 2 O HP, we evaluated the morphological change and gene expression level in cells immediately (HP 8 h + without HP 0 h) and after culturing without HP stimulation for another 24 h (HP 8 h + without HP 24 h) or 72 h (HP 8 h + without HP 72 h).

Cell morphological analysis
Cell morphology was observed under phase contrast microscopy.The aspect ratio was defined as the longitudinal cell axis (major axis) divided by the transverse cell axis (minor axis).The aspect ratio was calculated using Fiji software [16,17].Each data set consists of measurements of 100 cells.

Cell viability assays
Cells were seeded in 96-well culture plates at a density of 1 × 10 4 cells per well and cultured overnight.The cells were then treated with or without 100 cmH 2 O HP for 48 hours.After treatment, cell viability assays were performed using the Cell Proliferation Kit I (MTT) (#11465007001, Roche, Switzerland).Briefly, MTT was added and incubated for 4 hours.Then, the formation of formazan from MTT was stopped by adding solubilization solution, and the absorbance of formazan was measured at 570 nm using a microplate reader (iMark, Bio-Rad, USA).We used the optical density (OD) value of control cells as a normalization control (100%).

Immunofluorescence
Immunofluorescence for filamentous actin (F-actin) was performed by Alexa Fluor Plus 555 Phalloidin (#A30106, Thermo Fisher Scientific, USA) staining.Briefly, cells were fixed with 4% paraformaldehyde (#163-20145, Wako, Japan) for 10 minutes at room temperature.After washing with phosphate buffered saline (PBS), cells were permeabilized in 0.1% Triton X-100 (#X100, Sigma-Aldrich, USA) in PBS for 15 minutes.After washing with PBS, cells were incubated with Alexa Fluor Plus 555 Phalloidin at room temperature for 30 minutes in the dark.After washing with PBS, cells were incubated with NucBlue Fixed Cell ReadyProbes Reagent (DAPI, #R37606, Thermo Fisher Scientific) for 15 minutes.The immunofluorescence in cells was examined on a fluorescence microscope (BZ-X810, Keyence, Japan).

Statistical analysis
All data were presented as the mean ± SD.Two-tailed Student's t-test was used to compare continuous variables between two groups for normally distributed variables.Data between groups for normally distributed variables were compared by a one-way analysis of variance (ANOVA) followed by Tukey's test.Data between groups for non-normally distributed variables were compared by Kruskal-Wallis test followed by Dunn's test.A p-value less than 0.05 was considered significant.Data were analyzed with GraphPad Prism 9 (GraphPad Software, USA).

Short-term HP treatment induced mild changes in cell morphology and growth factor expression in HREpCs
HREpCs were subjected to 100 cmH 2 O HP for 8 h, and then cultured without HP for another 24 h or 72 h (Fig 1A).Under phase contrast microscopy, we noticed that cell morphology and aspect ratio had almost no change induced by short-term (8 h) HP treatment (Fig 1B and 1C).The aspect ratio was defined as the longitudinal cell axis (major axis) divided by the transverse cell axis (minor axis) (Fig 1C).
We next investigated the expression of growth factors in HREpCs after short-term HP treatment.The expression of CSF2 was not significantly changed by short-term HP treatment.However, the CSF2 expression in the HP-treated cells decreased gradually with time and resulted in a significantly lower level at 72 h after removing from HP treatment (p < 0.05, vs. control, Fig 2).The expression of TGFB1 and VEGFA were increased by short-term HP treatment

Long-term HP treatment induced severe changes in cell morphology and growth factor expression in HREpCs
We also investigated the long-term effect of HP treatment on HREpCs.The HP treatment period was extended to 48 h (Fig 3A).Under phase contrast microscopy, we observed some cells showing a round shape 48 h after HP treatment (Fig 3B).The cellular aspect ratio was increased by long-term (48 h) HP treatment, but returns to the basic level in 72 h after removing from HP treatment, suggesting a reversible elongation of the HP-treated cells (p < 0.05, vs. control, Fig 3C).An MTT assay indicated the cell viability was about 6% lower in HP-treated cells than control cells (p = 0.0391, Fig 3D), suggesting a slight but statistically significant effect on cell viability/metabolic activity.Staining cells with F-actin also showed that HP-treated cells presented elongated morphology compared to control cells (Fig 3E).
We next investigated the expression of growth factors in HREpC after long-term HP treatment.The expression of CSF2 and VEGFB was decreased in the HP-treated cells after removing from HP treatment (p < 0.05, vs. control, Fig 4).The expression of TGFB2 and PDGFB

Discussion
The physiological level of HP is known to regulate diverse cell fate and functions, including differentiation, migration, apoptosis, and proliferation [18].However, intense and persistent changes in HP to abnormal levels can damage cells [19].We have recently demonstrated that elevated HP induces profibrotic properties in hepatic stellate cells and atrial stromal cells [8,20] and affects biphasically lymphocyte activation via complex mechanisms [21].AKI is a frequent complication of obstructive uropathy, which confers a poor prognosis [1].In human unobstructed kidneys, the IRPs range from zero to a few cmH 2 O, while in chronic kidney obstruction, they range from 68 to 95.2 cmH 2 O [2].Therefore, we loaded HREpCs to 100 cmH2O HP to mimic an elevated HP condition by a pneumatic pressurizing system.
Ex vivo exposure of cells to an elevated HP condition has been widely studied [22,23].We found that HREpCs acquired an elongated morphology 48 h after 100 cmH 2 O HP treatment, but not 8 h after 100 cmH 2 O HP treatment, indicating long-term HP treatment may induce EMT in HREpCs.Moreover, the aspect ratio returns to the basic level 72 h after removing from HP treatment, suggesting elevated HP-induced elongation of HREpCs is reversible.In our previous study, we also found that the loading to 68 cmH 2 O HP for 24 h significantly changed the expression of EMT markers in HREpCs, such as vimentin, β-catenin, and α-SMA [12].EMT of renal epithelial cells can be involved in interstitial fibrosis [24].Therefore, biomechanical stresses, organ fibrogenesis, and EMT of tissue cells are likely linked to each other.
However, the effects of elevated HP-induced injury in renal epithelial cells remain unclear.It has been reported that growth factors regulate the pathogenesis of kidney disease and renal cell repair [25].In a rat model, the expression of epidermal growth factor (EGF) and transforming growth factor-beta (TGFB) was increased in the post-obstructed kidney [26].These authors suggest that EGF is involved in the promotion of recovery from obstructive uropathy.Meanwhile, TGFB is thought to play a major role in post-injured renal fibrosis, which might result in renal dysfunction [26].Another study, using a mouse model, found that hepatocyte growth factor (HGF) attenuates apoptosis in renal cells and reduces the progression of interstitial fibrosis caused by obstructive uropathy [27].In this study, our ex vivo data also showed that elevated HP disturbs the expression of growth factors in HREpCs, including CSF2, TGFB, platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF).
The role of CSF2 in kidney disease has not been well investigated to date.One study has recently reported that CSF2-mediated macrophage transition promotes renal epithelial cell repair after kidney injury [28].Our data showed that the expression of CSF2 did not immediately change after the HP treatment.However, the expression of CSF2 was decreased in the short-term HP-treated cells 72 h after removing from HP treatment.Similarly, the expression of CSF2 persistently decreased in the long-term HP-treated cells 24 h after removing from HP treatment.Elevated HP induced a down-regulated expression of CSF2, which may impair renal cell repair.
TGFB is widely accepted as a critical mediator of renal fibrosis [29].We have reported that ureteral obstruction increases the expression of TGFB in obstructed kidneys [14].The TGFB1, TGFB2, and TGFB3 isoforms are transcribed from different genes but have similar biological functions [29].Our results showed that expression of TGFB1 was increased after the shortterm HP treatment, but returned to the basic level in 24h after removing from HP treatment.The expression of TGFB2 was decreased by short-term HP treatment and returned to the basic level at 72h after removing from HP treatment.These results suggest that short-term HPinduced changes in TGFB expression are reversible.The expression of TGFB3 was increased in the long-term HP-treated cells after removing from HP treatment.However, the expression of TGFB2 was persistently decreased after loading to long-term HP.Meanwhile, TGFB also plays multiple physiological roles, such as cell proliferation, apoptosis, differentiation, autophagy, and immune responses [6].Clinical studies of anti-TGFB therapies in kidney disease have failed due to a lack of beneficial effects on renal injury [30].Thus, the role of TGFB in elevated HP-induced renal cell injury is conflicting and may depend on the type of TGFB isoform.
In the kidney, PDGF promotes the proliferation and recruitment of fibroblasts, mesangial cells, pericytes, and smooth muscle cells [31].PDGF is critically involved in the progression of kidney diseases, including mesangioproliferative glomerulonephritis, interstitial fibrosis, acute kidney injury, and diabetic nephropathy [25,31].PDGF signaling is highly involved in fibroblast transformation, and capillary damage that results in alterations in renal hemodynamics [25].We found that expression of PDGF was persistently decreased after loading to long-term HP.However, the role of PDGF in elevated HP-induced renal cell injury is unclear, and it may involve reciprocal interactions between other cells in the kidney.
VEGF stimulates glomerular endothelial cell and peri-tubular endothelial cell proliferation, migration, and survival [32].It has been reported that VEGF is a double-edged sword in renal disease.Early VEGF supplementation protects against renal injury, whereas late anti-VEGF treatment attenuates renal fibrosis progression [33].Moreover, VEGF inhibitors are commonly used in anti-angiogenic therapy of cancer, but these agents often cause renal dysfunction [34].Higher serum VEGF levels are positively correlated with renal dysfunction in patients with diabetic nephropathy [35].It has been suggested that the primary function of VEGF is to promote survival rather than angiogenesis [36].In our study, we found that the expression of VEGF was increased after the short-term HP treatment, but returned to the basic level in 24h after removing from HP treatment.However, the expression of VEGF decreased in the long-term HP-treated cells after removing from HP treatment.These results suggest that the long-term HP treatment, but not short-term HP treatment, impaired VEGF expression and may result in renal dysfunction.
It has been reported that mechanical stress-related diseases are associated with various signaling pathways, such as TGFB/SMAD signaling pathway, RhoA/rho-associated protein kinase (ROCK) signaling pathway, and Wnt/β-catenin signaling pathway, etc. [37].Pathological HP induces atrial fibroblast proliferation and collagen deposition through the TGFB/ SMAD pathway [38].Elevated HP activates the hepatic stellate cells to facilitate fibrosis progression via the cytoskeleton-related signals (i.e., RhoA, ROCK, and α-SMA) [8].Research on the molecular mechanisms of elevated HP-induced kidney injury is lacking.Our previous study found that the loading to 68 cmH2O HP for 24 h significantly upregulated the expression of β-catenin in HREpCs, indicating the Wnt/β-catenin signaling pathway may be one of the major signaling pathways involved in HP-induced kidney injury [12].
This study has several limitations.Firstly, due to genetic and phenotypic changes in immortalized cells, we used primary HREpCs for the experiment.As these primary cells have very limited proliferative property, it is difficult to expand enough cells to isolate proteins for additional experiments to further confirm our findings from real-time PCR.Secondly, the potential morphological alterations of HP-treated HREpCs have not been extensively studied and remain largely unknown.Thus, we will further explore the changes in cell ultrastructure using transmission electron microscopy.Thirdly, it remains unclear how HP changes the expression of growth factors in renal epithelial cells.Therefore, it is critical to elucidate the relevant molecular mechanism associated with renal cell injury in response to elevated HP by further experiments.Lastly, obstructive uropathy-related AKI is caused not only by renal epithelial cells, but also by the reciprocal interactions between stromal cells, endothelial cells, and immune cells.Otherwise, it is critical to elucidate the key growth factors associated with obstructive uropathy-related AKI by further co-culture experiments and in vivo animal studies.
In conclusion, our ex vivo investigations suggest that long-term exposure of renal epithelial cells to elevated HP induces reversible morphology changes.Moreover, elevated HP disturbs the expression of growth factors in renal epithelial cells (Fig 5).Targeting key growth factors may provide a novel therapeutic approach to elevated HP-induced kidney injury, such as obstructive uropathy.

Fig 5 .
Fig 5. Graphical representation of the significant alterations due to HP treatment in HREpCs.(A) Short-term HP treatment induced mild changes in cell morphology and growth factor expression in HREpCs.(B) Long-term HP treatment induced severe changes in cell morphology and growth factor expression in HREpCs.https://doi.org/10.1371/journal.pone.0310001.g005